Mycoplasma haemofelis (Haemobartonella felis) is a hemotropic pathogen that causes acute and chronic diseases in cats. Distributed worldwide, the parasite has a significant impact on the health and well being of this species. The disease in cats was first reported in the United States in 1953. Acute infection with M. haemofelis is associated with a massive parasitemia of red blood cells that leads to a severe and sometimes fatal hemolytic anemia. The parasite is also notorious for its ability to evade the immune response of the host and successfully establish chronic infection. Furthermore, despite an intense immune response and/or antibiotic treatment, cats often remain asymptomatic carriers following infection. M. haemofelis is recognized as a secondary pathogen in conjunction with retroviruses, including Feline Leukemia Virus (FeL V) and Feline Immunodeficiency Virus (FIV), and might promote neoplastic transformation of hematopoietic cells in these cats. Recent studies based on polymerase chain reaction testing (PCR) have shown that about 25% of all cats that are anemic and/or acutely ill have a M. haemofelis infection.
To provide a commercially viable immunoassay for the diagnosis of M. haemofelis, a convenient and renewable source of antigen is needed for developing an immunoassay, as well as one that can be standardized. Since M. haemofelis cannot be grown in culture, the only source of antigen for an immunoassay is whole parasites harvested from an infected cat. This is not a convenient source and preparations of whole cell or membrane antigens are difficult to standardize.
The identification of immunogenic proteins of pathogens is important for the development of serologic diagnostic assays. Two-dimensional polyacrylamide gel electrophoresis (SDSP AGE), followed by mass spectrometry and microsequencing is a commonly used method for identifying candidate proteins (Meens et al. 20006; Huntley et al., 2007, Delvecchio et al., 2006, Sellman et al., 2005; Jacobsen et al. 2005). However, low and differentially expressed antigens cannot be identified using this technique. Several groups have used Phage lambda vectors to construct genomic expression libraries of mycoplasmal pathogens. To overcome the uncommon usage of the opal stop codon (UGA) by some Mycoplasma spp. to encode tryptophan, expression libraries constructed in E. coli harboring an inducible opal suppressor may be used to improve the results achieved. Following induction, clones that are immunodominant can be identified by screening the library with convalescent-phase or immune sera. Recombinant antigens are convenient, renewable, and once purified, can be standardized for use in an immunoassay.